Biochemistry, Replication and Transcription

Introduction

Replication is the process that allows cells to regenerate or create new DNA sequences through multiple regulated steps. Replication is utilized by the cell during the S phase of the cell cycle in which new DNA synthesis occurs to prepare for the cell division. With the help of many different specific enzymes, DNA has the ability to copy itself in the nucleus of a cell through the process of replication. Both prokaryotic and eukaryotic cells need to replicate and therefore both go through a similar yet different method. Firstly prokaryotes have round DNA that is connected into a ring and therefore only has one location for replication to start. Eukaryotic cells are a bit different as they are organized into tight chromosomes around histones making them linear. Replication in known to be semiconservative because the original DNA (the parent strand) splits and combines with new bases (the daughter strand) to create a new double helix of genes.[1] Specific viruses are also able to hijack the replication mechanism of a cell and use transcription to create viral proteins and genetic information. Transcription is the process of creating new RNA by using the parent strands as a template to match the required ratios of cytosine (C), guanine(G), uracil (U) and adenine (A). This newly synthesized RNA then has to undergo a further post-transcriptional transformation to protect itself from degradation once it leaves the nucleus on its was to the ribosomes. Both replication and transcription are essential to maintaining homeostasis in the body and protecting against mutations that occur due to environmental stresses.

Fundamentals

Replication occurs in the cell nucleus and is the process of creating new DNA from nucleotides. Three main stages of replication occur during each creation of new DNA: initiation, elongation, and termination.

Although similar in many ways, DNA transcription is the process of DNA being turned into messenger RNA to generate proteins at the site of the ribosomes. Transcription also has a three-step process that is broken down into initiation, elongation, and termination, followed by some post-transcriptional modification in eukaryotic cells; this is the first step in gene expression and is the base for how all proteins are created and regulated.

Issues of Concern

Many issues can occur with replication or transcription, the most common being mutations leading to a loss of function. In replication, there are checkpoints after the S phase to ensure the production of the correct amount and quality of DNA. However, one major common issue is the loss of this regulation due to a mutation, resulting in the cell becoming cancerous.[2] Many mutations may go unnoticed as they are silent or uncovered by the regulatory mechanisms in the cell. Some mutations, however, can directly affect the expression of some genes, leading to the lack of an essential protein. This loss of function can lead to many different issues, some life-threatening depending on which cell is affected.

Molecular

For the DNA to undergo replication, it first must enter a less strained formation, which it is able to achieve through acetylating the histone to expose the backbone.[3][4] Once in this state, the DNA can begin replication with the assistance of specific proteins, the first being DNA helicase. The DNA helicase is in charge of distributing the hydrogen bonds between base pairs in the DNA and in this way splits the DNA strand into two single strand templates. The helicase often starts disconnecting the DNA in an area that has a high concentration adenine (A) and thymine (T). Since these bases have only two hydrogen bonds, instead of three, it is an ideal place for the helicase to start unwinding the DNA. Once the DNA begins splitting, a replication fork is formed allowing for a second protein called RNA primase, to initiate placing RNA bases complementary to the template strand bases. The DNA strands are directional with one side (3') denoted by a hydroxyl group and another represented by a phosphate group (5'). Once the RNA primase places a small RNA primer to the template strand, then the DNA polymerase is able to attach to those short segments and is able to place complementary bases along the template strand, beginning the elongation phase of DNA replication.[5] On the leading strand, the DNA polymerase continues in constant motion while the lagging strand has to be copied in short segments in the opposite direction since DNA polymerase only codes 5' to 3'. Due to the short repetitive nature of the of these lagging fragments, the DNA placed on these sections has the name "Okazaki fragments." For bacteria and other prokaryotes, DNA polymerase I, II, IV, and V are used for checking and repairing mistakes made by polymerase III. In eukaryotes, these polymerases are referred to as alpha, delta, and epsilon. In both cases, the complementary bases are placed, but the RNA primer must be eliminated and replaced with the proper DNA sequence. An enzyme called exonuclease is responsible for this action; removing the RNA primer and matching the template strand with necessary bases. This replacement begins the last step of replication known as termination. Upon replacement of the primer, another enzyme called DNA ligase connects the Okazaki fragments, created on the lagging strand. The DNA ligase is able to connect nucleotides through the creation of phosphodiester bonds.[6] Crating these bonds is the final process in DNA replication, leading to the formation of 2 semiconservative DNA strands. They are referred to as semiconservative because each double helix now consists of half the template strand and half new bases added. For the DNA to avoid harmful degeneration, telomerase is used to add telomeres to the end of the DNA sequence.

Transcription is carried out by the primary enzyme known as RNA polymerase, which is able to break the hydrogen bonds between the bases leading to a single-stranded template. The RNA polymerase must receive a signal to start creating RNA. In prokaryotes, the RNA polymerase is able to bind directly to a promoter region. This region is known as a TATA box due to the presence of high frequency of adenine and thymine. This process is slightly different in eukaryotes because aside from the promoter region the RNA polymerase needs a transcription factor to bind to the promoter first.[7] This process of the RNA polymerase getting into the proper position is known as initiation. Once the RNA polymerase is able to bind to the section of the gene that will undergo transcription, it continues to separate the double helix and place RNA in a 5' to 3' manner. The RNA polymerase places complementary bases to the template strand, except instead of placing thymine with every adenine, the polymerase places a new base called uracil (U).[8] This process is the elongation phase as the RNA polymerase continues down the template creating a new complementary single-stranded RNA. The elongation step proceeds until the polymerase meets a hairpin loop structure known as the termination sequence, which causes the polymerase to fall off, thus beginning the termination phase. Unlike DNA created from replication, this new RNA strand needs to travel from the nucleus out into the cytosol to create proteins through translation. In preparation for the RNA to move out of the nucleus, it must first undergo some significant post-transcriptional modification to avoid degradation. The single-stranded RNA receives a 5' capping by 7-methylguanosine, which is often an mRNA sequence, as well as the addition of poly-A-tail on the 3' end. Both of these modifications help to protect the RNA as it travels to the ribosomes. During these modifications, eukaryotic cells also undergo splicing, in which portions of the RNA called introns are cut out, leaving only the required bases for translation, known as exons.[9] Once prepared for travel the mRNA continues out of the nucleus for translation.

Testing

A Southern blot tests DNA replication, looking for specific sequences. This test checks for the presence of DNA and shows if replication has occurred in the cell. On the other hand, testing for RNA after transcription requires the use of a Northern blot.[10] The Northern blot can test the presence of RNA or mRNA and therefore examines gene expression.

Clinical Significance

DNA sequencing has become much more effective in the last few years, allowing for the possibility of gene therapy. Gene therapy is the process of changing DNA or transcription to express specific DNA or RNA more promptly.[11] Theoretically, by changing the gene expression, one can reverse mutations that may occur, thus limiting the detrimental effects of miscoded DNA.[12] Gene therapy is only in its embryonic stages, but with more research, it could be a significant cure for many diseases including autoimmune diseases. DNA is the building block of all organisms, making DNA replication a crucial process for life. The creation of RNA from transcription is equally important for larger multicellular organisms to create enzymes and proteins necessary for survival. Only through effective regulation of both processes will humans be able to carry out fundamental biological functions.

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